NuPotential will use SBIR funds to improve a patent pending method to reprogram cells using a novel small molecule library. Reprogrammed cells generated using this improved method will be referred to as NuPiPSsm" cells. In addition, NuPotential will partner with VistaGen, Inc., to differentiate NuPiPSsm" cells into cardiomyocytes and assess cardiomyocyte functionality. This proposal meets several objectives as outlined in PA-09-249 including: 1) the development of cell-based therapies and production of isogenic compatible cells for transplant;2) evaluation of a potentially safer reprogrammed cell type (referred to as NuPiPSsm" cells);3) assessing whether current ES optimized procedures for developing ES-derived cardiac cells can be applied to NuPiPS" and NuPiPSsm" cells (Current methods developed at VistaGene produce cultures of more than 50% cardiomyocytes and provide a good basis of transplantation cells at various stages of cardiac development.). The reprogrammed cell lines, differentiated cardiomyocytes and the protein microarrays generated from these SBIR funds will also be made commercially available for applications such as drug discovery and toxicity screening. Direct reprogramming of somatic cells to induced pluripotent stem (iPS) cells has been demonstrated by forced expression of four transcription factors (also referred to as iPS factors). These initial demonstrations have provided valuable insight into molecular mechanisms of somatic cell reprogramming and raised the possibility that alternative strategies could be developed on an industrial scale to produce pluripotent stem cells without using embryos. However, key challenges remain for reprogramming-based technologies to fully realize commercial potential. These include improving efficiencies and alleviating the dependence of viral vector transduction for the therapeutic applications of human regenerative medicine. Emerging information has indicated that activation of the endogenous somatic cell reprogramming factor transcription network is a path that addresses both issues. Consistent with results from other labs, NuPotential's preliminary data demonstrate that the endogenous somatic cell reprogramming factor network is active in pluripotent stem cells, but not active in human dermal fibroblasts. Differences between these cell types include pluripotent gene promoter methylation levels and histone modifications, including histone acetylation. Furthermore, our preliminary data indicates that reprogramming efficiency is significantly enhanced by introducing a single transcription factor (instead of the previously described four factors) in combination with specific knockdown of one or more epi-genes (e.g., DNMTs, HDACs) encoding repressive epigenetic regulators in adult human fibroblasts. Therefore, the proposed SBIR studies are designed to build on these findings and establish proof of principle that novel small molecules, designed and developed by NuPotential, Inc., can replace the lentiviral delivered shRNA component currently required to reprogram human dermal somatic cells to a pluripotent state and that the newly reprogrammed cells (referred to as NuPiPSsm" cells) are similar to human ES cells in their differentiation capacity and are sufficiently scalable for industrial applications. To demonstrate this differentiation potential, NuPotential will partner with VistaGen Therapeutics, Inc., one of the leaders in developing and providing stem cell-based systems for drug discovery and development, to compare differentiation in vitro of NuPiPS"(NuPotential's original reprogrammed cell type), NuPiPSsm", iPS and human ES cells into a variety of lineages, with a specific focus on cardiomyocytes. Current pluripotent transcription factor-induced somatic cell reprogramming technology (iPS technology) is associated with epigenetic modification of the promoter regions of pluripotent genes. NuPotential's proprietary somatic cell reprogramming platform is based on targeting the epigenome by inhibiting repressive regulatory components (e.g., DNMTs, HDACs) to induce pluripotency genes and restore differentiation potential. NuPotential has filed patents on numerous embodiments of these approaches and has recently been issued its foundation patent. To our knowledge this is the first issued patent claiming production of reprogrammed cells without using embryos or transgenes. Fulfillment of the Phase I aims will yield novel information about basic reprogramming mechanisms that will be applied to improving the efficiency of nuclear reprogramming by directly inhibiting specific repressive epigenetic regulatory components. In Phase II, NuPotential will use small molecules (derived from a proprietary library designed to contain novel and highly specific epi-drugs) to specifically inhibit repressive epigenetic regulatory targets identified and validated in Phase I to de-repress pluripotency gene expression and restore a pluripotent state to differentiated somatic cells in culture. Proven efficient, these small molecules can be used to improve livestock cloning, as well as to produce new reprogrammed pluripotent stem cells (RePSCs") for therapeutic applications without the use of transgenes, viral vectors, eggs, or embryos. PUBLIC HEALTH RELEVANCE: NuPotential will use SBIR funds to improve a patent pending method to reprogram cells using a novel small molecule library. Reprogrammed cells generated using this improved method will be referred to as as NuPiPSsm" cells. In addition, NuPotential will partner VistaGen, Inc. to differentiate NuPiPSsm" cells into cardiomyocytes and assess cardiomyocyte functionality. This proposal meets several objectives as outlined in PA-09-249 including: 1) the development of cell-based therapies and production of isogenic compatible cells for transplant; 2) evaluation of a potentially safer reprogrammed cell type (referred to as NuPiPSsm" cells);3) assessing whether current ES optimized procedures for developing ES-derived cardiac cells can be applied to NuPiPS" and NuPiPSsm" cells (Current methods developed at VistaGene produce cultures of more than 50% cardiomyocytes and provide a good basis of transplantation cells at various stages of cardiac development.). The reprogrammed cell lines, differentiated cardiomyocytes and the protein microarrays generated from these SBIR funds will be made commercially available for applications such as drug discovery and toxicity screening.